1. Field of the Invention
The invention relates to transdermal delivery of antigen by a liposome formulation to induce an antigen-specific immune response.
2. Description of the Related Art
Liposomes are smectic mesophases, which have been defined in the following manner by D. M. Small (Handbook of Lipid Research, Vol. 4, Plenum, N.Y., pp. 49-50): "When a given molecule is heated instead of melting directly into anisotropic lipid it may instead pass through intermediate states called mesophases or liquid crystals, characterized by residual order in some directions but by lack of order in others . . . In general, the molecules of liquid crystals are somewhat longer than they are wide and have a polar or aromatic part somewhere along the length of the molecule. The molecular shape and the polar-polar or aromatic interaction permit the molecules to align in a partial ordered array . . . These structures characteristically occur in molecules that possess a polar group at one end. Liquid crystals with long range order in the direction of the long axis of the molecule are called smectic, layered, or lamellar liquid crystals . . . in the smectic states, the molecules may be in single or double layers, normal or tilted to the plane of the layer, and with frozen or melted aliphatic chains."
As an example of work in the field of transdermal delivery of antigen, Paul et al. (1995) and Paul and Cevc (1995) (hereinafter "the Cevc lab") were not able to use liposomes for transdermal immunization. The Cevc lab used three different lipid formulations: mixed micelles, liposomes, and transfersomes in attempting to cause the transdermal delivery of antigen. Lipid was provided as an ethanol solution of soybean phosphatidylcholine (SPC); liposomes were formed by sonication, then freeze-thawed, and finally filtered for the purposes of sterilization and improved sample homogeneity. Mixed micelles contained SPC and bile salt (BS) in a mole ratio of 1:1, transferosomes contained SPC and BS in a mole ratio of 9:2, and liposomes contained SPC but no BS.
Because they contain a significant proportion of bile salts, mixed micelles and transferosomes cannot be considered liposomes (i.e., smectic mesophases) as stated by D. M. Small (Handbook of Lipid Research, Vol. 4, Plenum, N.Y., p. 95): "Class IIIA lipids . . . exhibit lyotropic mesomorphism at low water concentrations and form liquid crystals . . . At higher water concentrations, however, these liquid crystals dissolve to form micelles. Aliphatic molecules such as soaps, lysolecithin, and aliphatic detergents, are representative of class IIA lipids. In class IIIB lipids . . . bulky aromatic ring systems often comprise the hydrophobic component of the molecule. These compounds form micelles, but do not form liquid crystals. Molecules typical of this class are bile salts (e.g., Na cholate, Na deoxycholate, and Na chenodeoxycholate), saponins, and rosin soaps." As noted above in Small's definition of smectic mesophases, liposomes are a type of liquid crystals.
FIG. 1 of Paul et al. (1995) shows that only a formulation of antigen and transferosomes induced an immune response as measured by titer of antigen-specific antibody. Topically applied formulations of antigen in solution, antigen and mixed micelles, and antigen and liposomes (i.e., smectic mesophases) did not induce an immune response equivalent to that induced by subcutaneous injection. Therefore, there was a positive control (i.e., antigen and transfersomes) to validate their negative conclusion that a formulation of antigen and liposomes did not cause transdermal immunization.
Moreover, Paul and Cevc (1995) state on page 145, "Large molecules normally do not get across the intact mammalian skin. It is thus impossible to immunize epicutaneously with simple peptide or protein solutions." They conclude, "The dermally applied liposomal or mixed micellar immunogens are biologically as inactive as simple protein solutions, whether or not they are combined with the immunoadjuvant lipid A."
Despite the aforementioned contrary teaching, we have found that liposomes do provide a transdermal delivery system for antigen that can induce an antigen-specific immune response.